The detection and diagnosis of human conditions is of obvious importance for the treatment of disease. Numerous characteristics of diseases have been identified and many are used for their diagnosis. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes can include the expression of viral genes in infected cells, changes in the expression patterns of genes in affected cells, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases can be aided by the assessment of such cell states.
Routinely, for patients suspected of having one or more infectious diseases, for example human papilloma virus or herpes simplex virus, a sample of cells is taken from the patient for analysis. Generally, such a sample is in the form of a swipe or cellular scrape from the area primarily affected by the disease. These swipes usually collect a mixture of normal and diseased cells with a very limited total number of cells. The collected cells are traditionally smeared onto a slide for further analysis. When biochemical analysis was attempted, it was done at the expense of a cytological analysis and was done via qualitative methods such as in situ hybridization.
Routinely, the cervical sample obtained for conventional cytology is smeared onto a slide for morphological analysis. If this sample identifies potential disease by cell cytology, the patient must return for colposcopy to have a second sample collected for repeat cytology and/or genetic analysis and other molecular tests such DNA, RNA or protein. Recently, liquid cytology media have appeared on the market, which provide for enhanced morphology. These media were discovered to be amendable to molecular tests such as for HPV DNA, however, cells are routinely collected into 10-20 ml of preserving agents, which excessively dilute DNA, RNA and other asssayable biomolecules, making molecular testing less than ideal. Further, while the current preserving reagents preserve cellular morphology these reagents allow degradation of DNA and RNA, such that quantitative analysis becomes difficult or impossible upon storage.
Thus, present day analysis requires at least two samples to be obtained from a patient in order to determine cell morphology and quantitative genetic analysis. Current cytology methods use large volumes of a preserving agent which excessively dilute DNA, RNA and other assayable biomolecules. Further, while the current preserving reagents preserve cellular morphology these reagents allow degradation of individual biomolecules such as DNA, RNA and protein, such that quantitative analysis becomes impossible.
Designing methods and media to preserve cells or tissue for analysis by both morphological and biochemical analysis has proven problematic in the past. For example, fixing cells or tissue for morphological analysis compromised the ability to do biochemical analysis. Preserving cellular RNA was done at the expense of DNA and morphological analysis. Similarly, preserving cellular DNA was done at the expense of RNA and morphological analysis.
It is an object of the present invention to provide a general collection medium for identifying cellular morphology and quantitatively preserving DNA, RNA and other biomolecules from a single sample regardless of the disease to be detected.
It is another object of the present invention to provide a universal cell collection medium for preserving cell morphology and nucleic acids, lipids, carbohydrates, or proteins in a cell sample where limited numbers of cells are available in the sample.
It is another object of the present invention to provide compositions and methods for solution-based direct analysis of biomolecules of interest. The methods and compositions of this invention solve problems encountered in non-solution-based methods such as in situ hybridization or non-direct methods which require separation of the biomolecule of interest from other cellular components before analysis.